Overexpression of a fusion protein of 4-coumaroyl-CoA ligase and polyketide synthase for raspberry ketone production in Chlamydomonas reinhardtii / 生物工程学报

Raspberry ketones have important therapeutic properties such as anti-influenza and prevention of diabetes. In order to obtain raspberry ketone from Chlamydomonas reinhardtii, two enzymes catalyzing the last two steps of raspberry ketone synthesis, i.e. 4-coumaryl-CoA ligase (4CL) and polyketide synthase (PKS1), were fused using a glycine-serine-glycine (GSG) tripeptide linker to construct an expression vector pChla-4CL-PKS1. The fusion gene 4CL-PKS1 driven by a PSAD promoter was transformed into a wild-type (CC125) and a cell wall-deficient C. reinhardtii (CC425) by electroporation. The results showed the recombinant C. reinhardtii strain CC125 and CC425 with 4CL-PKS1 produced raspberry ketone at a level of 6.7 μg/g (fresh weight) and 5.9 μg/g (fresh weight), respectively, both were higher than that of the native raspberry ketone producing plants (2-4 μg/g).

Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase deficiency: a case report and literature review / 中国当代儿科杂志

Mitochondrial 3-hydroxy-3-methylglutaryl CoA synthase deficiency (HMCSD) is caused by HMGCS2 gene mutation. This paper reports the clinical and genetic features of an infant with this disease. The 8-month-old female infant was admitted to the hospital with diarrhea for 1 week and fever and convulsion for 1 day. The child presented with seizures, acidosis, hypoglycemia, abnormal liver function, myocardial injury and coagulation dysfunction. The new homozygous mutation c.1502G>A(p.R501Q) in the HMGCS2 gene was found in the infant by genetic testing. The mutant gene was found to be harmful by bioinformatics software analysis. Urine organic acid analysis indicated that 4-hydroxy-6-methyl-2-pyranone was significantly increased, which was consistent with the results of genetic testing. The infant was definitely diagnosed with HMCSD.

Anti-diabetic activities of catalpol in db/db mice

The objective was to investigate the hypoglycemic action of catalpol in spontaneous diabetes db/db mice. 40 db/db mice were randomly divided into fi ve groups: model control gourp; db/db plus catalpol 40, 80, 120 mg/kg body wt. groups and db/db plus metformin 250 mg/kg group. Age-matched db/m mice were selected as normal control group. The mice were administered with corresponding drugs or solvent by gavage for 4 weeks. The oral glucose tolerance test was carried out at the end of 3rd week. After 4 weeks of treatment, the concentrations of fasting blood glucose (FBG), glycated serum protein (GSP), insulin (INS), triglyceride (TG), total cholesterol (TC) and adiponection (APN) in serum were detected. The protein expressions of phosphorylation-AMPKalpha1/2 in liver, phosphorylation-AMPKalpha1/2 and glucose transporter-4 (GLUT-4) in skeletal muscle and adipose tissues were detected by western blot. Real time RT-PCR was used to detect the mRNA expressions of acetyl-CoA carboxylase (ACC) and Hydroxymethyl glutaric acid acyl CoA reductase (HMGCR) in liver. Our results showed that catalpol could significantly improve the insulin resistance, decrease the serum concentrations of INS, GSP, TG, and TC. The concentrations of APN in serum, the protein expression of phosphorylation-AMPKalpha1/2 in liver, phosphorylation-AMPKalpha1/2 and GLUT-4 in peripheral tissue were increased. Catalpol could also down regulate the mRNA expressions of ACC and HMGCR in liver. In conclusion, catalpol ameliorates diabetes in db/db mice. It has benefi t eff ects against lipid/glucose metabolism disorder and insulin resistance. The mechanism may be related to up-regulating the expression of phosphorylation-AMPKalpha1/2.

Effect of high-fat diet on cholesterol metabolism in rats and its association with Na⁺/K⁺-ATPase/Src/pERK signaling pathway / 华中科技大学学报（医学）（英德文版）

Abnormal cholesterol metabolism is associated with an elevated risk of developing atherosclerosis, hypertension, and diabetes etc. Na(+)/K(+)-ATPase was found to regulate cholesterol synthesis, distribution and trafficking. This study aimed to examine the effect of high-fat diet on cholesterol metabolism in rats and the role of Na(+)/K(+)-ATPase/Src/ERK signaling pathway in the process. Forty male SD rats were evenly divided into high-fat diet group and control group at random. Animals in the former group were fed on high-fat diet for 12 weeks, and those fed on basic diet served as control. Blood lipids, including total cholesterol (TC), triglyceride (TG), high density lipoprotein-cholesterol (HDL-C), and low density lipoprotein-cholesteral (LDL-C) levels, were detected at 3, 6 and 12 weeks. The ratio of cholesterol content in cytoplasm to that in cell membrane was detected in liver tissues. RT-PCR and Western blotting were used to measure the expression of lipid metabolism-associated genes (HMG-CoA reductase and SREBP-2) after 12-week high-fat diet. Na(+)/K(+)-ATPase/Src/ERK signaling pathway-related components (Na(+)/K(+)-ATPase α1, Src-PY418 and pERK1/2) were also measured by Western blotting. The results showed that the serum TC, TG, and LDL-C levels were significantly higher in high-fat diet group than those in control group, while the HDL-C level was significantly lower in high-fat diet group at 6 weeks (P<0.01). High-fat diet led to an increase in the cholesterol content in the cytoplasm and cell membrane. The ratio of cholesterol content in cytoplasm to that in cell membrane was elevated over time. The expression of HMG-CoA reductase and SREBP-2 was significantly suppressed at mRNA and protein levels after 12-week high-fat diet (P<0.05). Moreover, high-fat diet promoted the expression of Na(+)/K(+)-ATPase α1 but suppressed the phosphorylation of Src-PY418 and ERK1/2 at 12 weeks (P<0.05). It was concluded that high-fat diet regulates cholesterol metabolism, and Na(+)/K(+)-ATPase signaling pathway is involved in the process possibly by regulating the expression of lipid metabolism-associated proteins HMG-CoA reductase and SREBP-2.

"Nitrate stimulating effect" in Amycolatopsis mediterranei--from discovery to mechanistic studies / 生物工程学报

Nitrate not only remarkably stimulates the rifamycinbiosynthesis in Amycolatopsis mediterranei, but also influences the primary metabolisms, including the inhibition of fatty acids biosynthesis in the bacterial. This phenomenon has been designated as "Nitrate Stimulating Effect" by the late Prof. J.S. Chiaosince its discovery in the 1970's, and has been found in many other antibiotics-producing actinomycetes subsequently. Based on the research in his laboratory, we have revealed that the nitrate stimulation effect mainly manifests in two aspects over the last two decades. First, nitrate promotes the supply of rifamycin precursors, e.g., UDP-glucose, AHBA, malonyl-CoA and methylmalonyl-CoA. Specifically, the biosynthesis of fatty acids is inhibited by nitrate consequently the acetyl-CoA is shunted into malonyl-CoA. Second, nitrate facilitates the expression of genes in the rifclulsterthat encodes rifamycin biosynthetic enzymes. Following our current understanding, the future research will focus on the signals, the signal transduction pathway and the molecular mechanisms that dictate nitrate-mediated transcriptional and post-translational regulations.

Preliminary study on efficacy and mechanism of Atractylodes Macrocephelae Rhizoma extracts in metabolic hyperlipidemia rats / 中国中药杂志

Hyperlipidemia is a major factor causing coronary heart disease and atherosclerosis. The high-density lipoprotein cholesterol (HDL-C) is a major indicator for measuring lipid levels. However, there is no an effective medicine that can obviously increase HDL-C at present. According to previous laboratory studies, atractylodes macrocephalae extracts could significantly increase HDL-C level. In this study, the metabolic hyperlipidemia rat model was established by feeding high-sugar and fat diets and alcohol-drinking to explore the effect and mechanism of atractylodes macrocephalae extracts on hyperlipidemia rats. According to the findingins, different doses of atractylodes macrocephalae extracts could reduce the levels of TC, TG, LDL-C, ACAT and increase the contents of LCAT, HDL-C. Particularly, the atractylodes macrocephalae extracts (100 mg · kg(-1) group showed increase in HDL-C by about 50% and significant declines in HMG-CoA reductase, TC, TG. In conclusion, Atractylodes Macrocephelae Rhizoma extracts could effectively regulate the dyslipidemia of hyperlipidemia rats, especially on HDL-C. Its mechanism may be related to reduction in cholesterol synthesis by inhibiting HMG-CoA reductase in livers and increase in lipid metabolism and transport by regulating LCAT and ACAT levels.

Estudio de la deficiencia de acil-CoA deshidrogenasa de cadena muy larga con hipoglicemia / Study of very long chain acyl-CoA deshydrogenase with hypoglycemia / Estudo da deficiência de acil-CoA desidrogenase de cadeia muito longa com hipoglicemia

La acil-CoA deshidrogenasa de cadena muy larga (VLCAD, very long chain acyl-Co A dehydrogenase) es considerada una enzima limitante en el sistema de beta-oxidación de ácidos grasos de cadena muy larga. Con base en la clínica presentada en esta deficiencia se han informado 3 fenotipos: VLCAD-C por presentar cardiomiopatía dilatada, VLCAD-H caracterizada por episodios de hipoglicemia y VLCAD-M en la cual se presentan episodios de miopatía con rabdomiólisis. Los estudios in vitro, que buscan intermediarios de la degradación mitocondrial de ácidos grasos, facilitan el diagnóstico de alteraciones hereditarias o adquiridas de la actividad de esta enzima. En el presente trabajo se analiza la producción de metabolitos en fibroblastos de pacientes con deficiencia de acil-CoA deshidrogenasa de cadena muy larga con hipoglicemia (VLCAD-H), incubados con ácido oleico deuterado. Se incubaron fibroblastos de cinco pacientes con deficiencia de acil-CoA deshidrogenasa de cadena muy larga (VLCAD) con hipoglicemia y de diez controles en presencia de ácido oleico deuterado. Se encontró un perfil característico luego de la incubación de fibroblastos con esta deficiencia. Los valores de D²C12:1 fueron similares entre los pacientes y los controles, mientras que los valores de D²C14:1 y D²C16:1 fueron encontrados significativamente más elevados en los fibroblastos de los pacientes. Este sustrato podría ser usado para realizar diagnóstico in vitro de la deficiencia de VLCAD-H.

Very-long-chain acyl-CoA dehydrogenase (VLCAD) is considered a rate-limit-ing enzyme in a very-long-chain fatty acid beta-oxidation system. Based on clinical presentation three phenotypes have been reported for this deficien-cy, as follows: VLCAD-C presenting cardiomiopathy, VLCAD-H characterized by episodes of hypoglycemia, and VLCAD-M including episodes of myopathy with rhabdomyolysis. In vitro studies, searching for intermediates of mitochondrial fatty acid degradation, are a tool for diagnosis of inherited or acquired alterations of this enzyme activity. The present work analizes the metabolite production in fbroblasts from patientes with VLCAD-H deficiency, incubated with deuterated oleic acid. Fibroblasts from ten controls and fve patients with VLCAD deficiency with hypoglycemia (VLCAD-H) were incubated in the presence of deuterated oleic acid. A characteristic profle was found after incuba-tion of fbroblasts with this deficiency. D²C12:1 values were similar between patients and controls, while values of D²C14:1 and D²C16:1 were significantly higher in the fbroblasts of patients. Therefore, this substrate could be used for in vitro diagnosis of VLCAD-H deficiency.

A acil-CoA desidrogenase de cadeia muito longa (VLCAD, very long chain acyl-Co A dehydrogenase) é considerada uma enzima limitante no sistema de beta-oxidação de ácidos graxos de cadeia muito longa. Com base na clínica apresentada nesta deficiência, foram informados três fenótipos: VLCAD-C por apre-sentar cardiomiopatia dilatada, VLCAD-H caracterizada por episódios de hipoglicemia e VLCAD-M em que ocorrem episódios de miopatia com rabdomiólise. Os estudos in vitro, em busca de intermediários da degradação mitocondrial de ácidos graxos, são uma ferramenta para o diagnóstico de alterações here-ditárias ou adquiridas da atividade desta enzima. O presente trabalho analisa a produção de metabólitos em fibroblastos de pacientes com deficiência de acil-CoA desidrogenase de cadeia muito longa, com hipoglicemia (VLCAD-H), incubadas com ácido oleico deuterado. Foram incubados fibroblastos de cinco pacientes com deficiência de acil-CoA desidrogenase de cadeia muito longa (VLCAD) com hipoglicemia, e de dez controles em presença de ácido oleico deuterado. Foi achado um perfil característico após a incubação de fibroblastos com esta deficiência. Os valores D²C12:1 foram semelhantes entre os pacientes e os controles, ao passo que os valores de D2C14:1 e D²C16:1 foram significativamente mais elevados nos fibroblastos dos pacientes. Este substrato poderia ser utilizado para realizar diagnóstico in vitro da deficiência VLCAD-H.

Study on lipid-lowering traditional Chinese medicines based on pharmacophore technology and patent retrieval / 中国中药杂志

The combined application of statins that inhibit HMG-CoA reductase and fibrates that activate PPAR-α can produce a better lipid-lowering effect than the simple application, but with stronger adverse reactions at the same time. In the treatment of hyperlipidemia, the combined administration of TCMs and HMG-CoA reductase inhibitor in treating hyperlipidemia shows stable efficacy and less adverse reactions, and provides a new option for the combined application of drugs. In this article, the pharmacophore technology was used to search chemical components of TCMs, trace their source herbs, and determine the potential common TCMs that could activate PPAR-α. Because there is no hyperlipidemia-related medication reference in modern TCM classics, to ensure the high safety and efficacy of all selected TCMs, we selected TCMs that are proved to be combined with statins in the World Traditional/Natural Medicine Patent Database, analyzed corresponding drugs in pharmacophore results based on that, and finally obtained common TCMs that can be applied in PPAR-α and combined with statins. Specifically, the pharmacophore model was based on eight receptor-ligand complexes of PPAR-α. The Receptor-Ligand Pharmacophore Generation module in the DS program was used to build the model, optimize with the Screen Library module, and get the best sub-pharmacophore, which consisted of two hydrogen bond acceptor, three hydrophobic groups and 19 excluded volumes, with the identification effectiveness index value N of 2. 82 and the comprehensive evaluation index CAI value of 1. 84. The model was used to screen the TCMD database, hit 5,235 kinds of chemical components and 1 193 natural animals and plants, and finally determine 62 TCMs. Through patent retrieval, we found 38 TCMs; After comparing with the virtual screening results, we finally got seven TCMs.

Chronic intermittent form of isovaleric aciduria in a 2-year-old boy / 소아과

Isovaleric aciduria (IVA) is caused by an autosomal recessive deficiency of isovaleryl-CoA dehydrogenase (IVD). IVA presents either in the neonatal period as an acute episode of fulminant metabolic acidosis, which may lead to coma or death, or later as a "chronic intermittent form" that is associated with developmental delays, with or without recurrent acidotic episodes during periods of stress, such as infections. Here, we report the case of a 2-year old boy with IVA who presented with the chronic intermittent form. He was admitted to Asan Medical Center Children's Hospital with recurrent vomiting. Metabolic acidosis, hyperammonemia, elevated serum lactate and isovalerylcarnitine levels, and markedly increased urine isovalerylglycine concentration were noted. Sequence analysis of the IVD gene in the patient revealed the novel compound mutations-a missense mutation, c.986T>C (p.Met329Thr) and a frameshift mutation, c.1083del (p.Ile361fs*11). Following stabilization during the acute phase, the patient has remained in a stable condition on a low-leucine diet.

Dietary carnosic acid suppresses hepatic steatosis formation via regulation of hepatic fatty acid metabolism in high-fat diet-fed mice

In this study, we examined the hepatic anti-steatosis activity of carnosic acid (CA), a phenolic compound of rosemary (Rosmarinus officinalis) leaves, as well as its possible mechanism of action, in a high-fat diet (HFD)-fed mice model. Mice were fed a HFD, or a HFD supplemented with 0.01% (w/w) CA or 0.02% (w/w) CA, for a period of 12 weeks, after which changes in body weight, blood lipid profiles, and fatty acid mechanism markers were evaluated. The 0.02% (w/w) CA diet resulted in a marked decline in steatosis grade, as well as in homeostasis model assessment of insulin resistance (HOMA-IR) index values, intraperitoneal glucose tolerance test (IGTT) results, body weight gain, liver weight, and blood lipid levels (P < 0.05). The expression level of hepatic lipogenic genes, such as sterol regulating element binding protein-1c (SREBP-1c), liver-fatty acid binding protein (L-FABP), stearoyl-CoA desaturase 1 (SCD1), and fatty acid synthase (FAS), was significantly lower in mice fed 0.01% (w/w) CA and 0.02% (w/w) CA diets than that in the HFD group; on the other hand, the expression level of beta-oxidation-related genes, such as peroxisome proliferator-activated receptor alpha (PPAR-alpha), carnitine palmitoyltransferase 1 (CPT-1), and acyl-CoA oxidase (ACO), was higher in mice fed a 0.02% (w/w) CA diet, than that in the HFD group (P < 0.05). In addition, the hepatic content of palmitic acid (C16:0), palmitoleic acid (C16:1), and oleic acid (C18:1) was significantly lower in mice fed the 0.02% (w/w) CA diet than that in the HFD group (P < 0.05). These results suggest that orally administered CA suppressed HFD-induced hepatic steatosis and fatty liver-related metabolic disorders through decrease of de novo lipogenesis and fatty acid elongation and increase of fatty acid beta-oxidation in mice.

Intractable metabolic acidosis in a child with propionic acidemia undergoing liver transplantation: a case report / 대한마취과학회지

Propionic acidemia (PA) is a rare autosomal recessive disorder of metabolism caused by deficient activity of the mitochondrial enzyme propionyl-CoA carboxylase. The clinical manifestations are metabolic acidosis, poor feeding, lethargy, vomiting, osteoporosis, neurological dysfunction, pancytopenia, developmental retardation and cardiomyopathy. Liver transplantation has recently been considered as one of the treatment options for patients with PA. This case report describes several anesthetic considerations for patients with PA undergoing liver transplantation. Understanding the patient's status and avoiding events that may precipitate metabolic acidosis are important for anesthetic management of patients with PA. In conclusion, anesthesia should be focused on minimizing the severity of metabolic acidosis with following considerations: (1) maintaining optimal tissue perfusion by avoiding hypotension, (2) preventing hypoglycemia, and (3) providing bicarbonate to compensate for the acidosis.

Mutation analysis of a family with 2-Methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency / 中华儿科杂志

<p><b>OBJECTIVE</b>The aim of this study was to explore the genetic features of a family with 2-methyl-3-hydroxybutyryl-CoA dehydrogenase deficiency (MHBDD) which may provide the basis for the diagnosis and genetic counseling.</p><p><b>METHOD</b>Clinical data of the proband was collected, total RNA and genomic DNA were extracted from the peripheral blood. The whole coding region of the ACAT1 gene was amplified by RT-PCR. 5' noncoding region of the ACAT1 gene and all 6 exons and flanking intron regions of the HADH2 gene were amplified by PCR. All amplification products were directly sequenced and compared with the reference sequence.</p><p><b>RESULT</b>(1) The patient was a one-year-old boy who presented with psychomotor retardation and astasia when he was admitted to the hospital. Biochemical test revealed slight hyperlactatemia (3.19 mmol/L) and magnetic resonance imaging showed delayed myelination. 2-Methylacetoacetyl-CoA thiolase deficiency was suggested by gas chromatography-mass spectrometry. (2) There was no mutation in the ACAT1 gene and a hemizygous missense mutation c.388C > T was found in the 4 exon of the HADH2 gene which resulted in p. R130C. Proband's mother was the heterozygote and the father was normal.</p><p><b>CONCLUSION</b>This is the first report on MHBDD patient and HADH2 mutation in China. p.R130C is responsible for the pathogenesis of the disease in the infant.</p>

Neuroprotective effects of L-carnitine against oxygen-glucose deprivation in rat primary cortical neurons / 소아과

PURPOSE: Hypoxic-ischemic encephalopathy is an important cause of neonatal mortality, as this brain injury disrupts normal mitochondrial respiratory activity. Carnitine plays an essential role in mitochondrial fatty acid transport and modulates excess acyl coenzyme A levels. In this study, we investigated whether treatment of primary cultures of rat cortical neurons with L-carnitine was able to prevent neurotoxicity resulting from oxygen-glucose deprivation (OGD). METHODS: Cortical neurons were prepared from Sprague-Dawley rat embryos. L-Carnitine was applied to cultures just prior to OGD and subsequent reoxygenation. The numbers of cells that stained with acridine orange (AO) and propidium iodide (PI) were counted, and lactate dehydrogenase (LDH) activity and reactive oxygen species (ROS) levels were measured. The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay and the terminal uridine deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling assay were performed to evaluate the effect of L-carnitine (1 microM, 10 microM, and 100 microM) on OGD-induced neurotoxicity. RESULTS: Treatment of primary cultures of rat cortical neurons with L-carnitine significantly reduced cell necrosis and prevented apoptosis after OGD. L-Carnitine application significantly reduced the number of cells that died, as assessed by the PI/AO ratio, and also reduced ROS release in the OGD groups treated with 10 microM and 100 microM of L-carnitine compared with the untreated OGD group (P<0.05). The application of L-carnitine at 100 microM significantly decreased cytotoxicity, LDH release, and inhibited apoptosis compared to the untreated OGD group (P<0.05). CONCLUSION: L-Carnitine has neuroprotective benefits against OGD in rat primary cortical neurons in vitro.

The Effects of Combined Treatment with an HMG-CoA Reductase Inhibitor and PPARgamma Agonist on the Activation of Rat Pancreatic Stellate Cells

BACKGROUND/AIMS: Hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase inhibitors (statins) and peroxisome proliferator-activated receptor gamma (PPARgamma) ligands can modulate cellular differentiation, proliferation, and apoptosis through various pathways. It has been shown that HMG-CoA reductase inhibitors and PPARgamma agonists separately inhibit pancreatic stellate cell (PaSC) activation. We studied the effects of a combination of both types of drugs on activated PaSCs via platelet-derived growth factor (PDGF), which has not previously been reported. The present study was performed to elucidate the underlying mechanisms of these effects by focusing on the impact of the signaling associated with cell-cycle progression. METHODS: Primary cultures of rat PaSCs were exposed to simvastatin and troglitazone. Proliferation was quantified using the BrdU method, and cell-cycle analysis was performed using a fluorescent activated cell sorter. The protein expression levels of smooth muscle actin (SMA), extracellular signal-regulated kinase (ERK), and a cell cycle machinery protein (p27Kip1) were investigated using Western blot analysis. RESULTS: Simvastatin reversed the effects of PDGF on cell proliferation in a dose-dependent manner. The combination of a low concentration of simvastatin (1 mM) and troglitazone (10 mM) synergistically reversed the effects of PDGF on cell proliferation but had no effect on cell viability. The expression of a-SMA was markedly attenuated by combining the two drugs, which blocked the cell cycle beyond the G0/G1 phase by reducing the levels of phosphorylated ERK and reversed the expression of p27Kip1 interrupted by PDGF. CONCLUSIONS: Simvastatin and troglitazone synergistically inhibited cell proliferation in activated PaSCs by blocking the cell cycle beyond the G0/G1 phase. This inhibition was due to the synergistic modulation of the ERK pathway and the cell cycle machinery protein p27Kip1.

Implementación diagnóstica de la deficiencia de acil-CoA deshidrogenasa de cadena muy larga / Diagnostic implementation of very long chain acyl-CoA dehydrogenase deficiency / Implementação diagnóstica da deficiência de acil-CoA desidrogenase de cadeia muito longa

La enzima acil-CoA deshidrogenasa de cadena muy larga (VLCAD) es un homodímero que cataliza la reacción inicial de la ß-oxidación mitocondrial de los ácidos grasos. El presente estudio tuvo como objetivo el análisis de los metabolitos producidos por fibroblastos incubados en presencia de sustratos tritiados vs. deuterados, como herramienta diagnóstica de la deficiencia de VLCAD. Fue encontrada severamente deprimida la oxidación de los sustratos tritiados en los fibroblastos de pacientes con esta enfermedad, asimismo la incubación con sustratos deuterados aportó un perfil característico en esta deficiencia. El método de valoración de agua tritiada, aunque inespecífico, debido a que la oxidación de sustratos tritiados también puede estar deprimida en otras deficiencias, es un buen método para sugerir una deficiencia de acil- CoA deshidrogenasa de cadena muy larga, si el análisis se compara con otros hallazgos propios de la deficiencia enzimática. Sin embargo, la determinación de los metabolitos deuterados es más específica, por encontrarse un perfil característico que muestra niveles elevados de los ácidos grasos octanoico, decanoico, dodecenoico, dodecanoico, tetradecenoico, tetradecanoico y hexadecenoico, lo que la hace diferente de otras deficiencias enzimáticas.

Very long-chain acyl-CoA dehydrogenase (VLCAD) is a homodimer that catalyzes the initial reaction of fatty acid ß-oxidation. The objective of the present study was to analyse the metabolites produced by fibroblasts incubated with tritiated vs. deuterated substrates, as a diagnostic tool for the diagnosis of VLCAD deficiency. A severe depression for oxidizing the tritiated substrates was observed for these patients' fibroblasts, and a characteristic profile for this deficiency was found when incubating fibroblasts with deuterated substrates. The method which evaluates the production of tritiated water is nonspecific as the oxidation of tritiated substrates can be found depressed in other fatty acid ß-oxidation disorders; however this method can suggest VLCAD deficiency if the tritiated water measurement is compared with others findings related to this deficiency. On the other hand the measurement of deuterated metabolites is more specific as a characteristic profile was found for this deficiency showing increased levels of the following organic acids: octanoic, decanoic, dodecenoic, dodecanoic, tetradecenoic, tetradecanoic and hexadecanoic, which is different from other fatty acid ß-oxidation disorders.

A enzima acil-CoA desidrogenase de cadeia muito longa (VLCAD) é um homodímero que catalisa a reação inicial da ß-oxidação mitocondrial dos ácidos graxos. O presente estudo teve como objetivo a análise dos metabólitos produzidos por Ibroblastos incubados em presença de substratos tritiados vs. deuterados, como ferramenta de diagnóstico da deIciência de VLCAD. Foi encontrada severamente deprimida a oxidação dos substratos tritiados nos Ibroblastos de pacientes com esta doença, do mesmo modo a incubação com substratos deuterados deu um perIl característico nesta deIciência. O método de avaliação de água tritiada, embora não especíIco, devido a que a oxidação de substratos tritiados também pode estar deprimida em outras deIciências, é um bom método para sugerir uma deIciência de acil-CoA desidrogenase de cadeia muito longa, se a análise é comparada com outros achados próprios da deIciência enzimática. Entretanto, a determinação dos metabólitos deuterados é mais especiIca, devido a que se encontra um perIl característico que mostra níveis elevados dos ácidos graxos octanoico, decanoico, dodecenoico, dodecanoico, tetradecenoico, tetradecanoico e hexadecenoico, o que a torna diferente de outras deIciências enzimáticas.

HMG CoA Reductase Inhibitors Inhibit HCV RNA Replication of HCV Genotype 1b but Not 2a

Replication of hepatitis C virus (HCV) is regulated by statin, one of 3-hydroxy-3-methylglutaryl CoA reducatase (HMG CoA reductase) inhibitors that block mevalonate pathway and cholesterol biosyntheis, which has been used usefully for health improvement and disease control in clinic. In order to know which statin can be used to inhibit HCV replication, we examined the effects of HCV genotype 1b replication by 6 kinds of statins with different structure. We treated six statins to HCV genotype 1b replicon cell. Atorvastatin, simvastatin, fluvastatin, mevastatin, and lovastatin inhibited HCV RNA replication and HCV protein expression in HCV genotype 1b replicon cells, though pravastatin did not affect HCV replication. In order to know whether inhibition of HCV replication by statin is depended on HCV genotype, we treated the statins to HCV genotype 2a producing cells, and investigated HCV RNA replication and HCV protein expression. HCV RNA replication and protein expression was not affected in HCV genotype 2a producing cells by treatment of statins and cholesterol inhibitor. These results suggest that HMG-CoA reductase and cholesterol inhibitors might be used depending on HCV genotype. In addition, inhibition of HCV genotype 1b replication by statins has been depended on structure of various statins which should be seriously selected for HCV clinic. In future, we will study on inhibition of another HCV genotype replication by HMG-CoA reductase and cholesterol inhibitors.

Levels of carnitine and acylcarnitines in reconstituted red blood cell samples washed with different concentrations of saline solutions / Niveles de carnitina y acilcarnitinas en muestras de glóbulos rojos reconstituidos y lavados con solución salina a diferentes concentraciones

Objective: To evaluate the percentage of carnitine and acylcarnitines remaining in red blood cells after washing them with different concentrations of saline solution. Materials and methods: Human blood samples were centrifuged and the blood cells were washed with different saline solutions. The final pellet was resuspended in PBS for card preparation and tandem mass spectrometry analysis. Results: It was found that carnitine, as well as short-chain, medium-chain, and long-chain acylcarnitines remain in red blood cells at average percentages of 19.3; 34; 34; and 32%, respectively. Significant differences were found for carnitine and acylcarnitine levels in blood washed with an isotonic solution compared to their levels using several hypotonic solutions (p<0.05). Conclusion: Because carnitine and acylcarnitines remained associated with the blood cells, we recommend using whole blood to measure these metabolites.

Objetivo: Evaluar el porcentaje de carnitina y acilcarnitinas que permanece en los glóbulos rojos, luego de lavarlos con solución salina a diferentes concentraciones. Materiales y métodos: Muestras de sangre humana fueron centrifugadas y los glóbulos rojos fueron lavados con solución salina a diferentes concentraciones. El pellet obtenido, fue resuspendido en PBS para preparación de tarjetas y análisis por espectrometría de masa en tandem. Resultados: La carnitina, así como las acilcarnitinas de cadena corta, cadena media y cadena larga, permanecen en los glóbulos rojos a porcentajes promedio de 19,3; 34; 34; and 32%, respectivamente. Se encontró diferencia significativa al comparar los niveles de carnitina y acilcarnitinas de sangre lavada con una solución salina isotónica vs. varias hipotónicas (p<0.05). Conclusión: debido a que la carnitina y las acilcarnitinas permanecen en los glóbulos rojos, se recomienda el uso de sangre entera para medir los niveles de estos metabolitos.

Diagnóstico "in vitro" de la deficiencia de ACIL-CoA deshidrogenasa de cadena corta / \"In vitro\" diagnosis of short chain acyl-CoA dehydrogenase deficiency

La acil-CoA deshidrogenasa de cadena corta (SCAD) cataliza la primera reacción de la degradación de ácidos grasos de 4 a 6 átomos de carbono. Su deficiencia debe ser siempre confirmada por estudios de laboratorio. En el presente trabajo, fueron incubados fibroblastos de pacientes que presentaban la deficiencia de SCAD, en presencia de sustratos tritiados. Fue encontrada diferencia significativa (p<0,05) al comparar la degradación de palmitato y miristato tritiado entre controles y pacientes con deficiencia de SCAD.

Short-chain acyl-CoA dehydrogenase (SCAD) is the key enzyme for degrading fatty acids with a 4-6 atoms carbon chain. It is necessary to always confirm this deficiency using laboratory methods. Fibroblasts of patients suffering SCAD deficiency were incubated with tritiated palmitate and miristatesubstrates. A significant difference (p<0.05) was found when comparing tritiated palmitate and miristate degradation between controls and patients suffering SCAD deficiency.

Mitochondrial Aldehyde Dehydrogenase Precursor Protein and 3-hydroxy-3-methylglutaryl-coenzyme A synthase 2: Two-dimensional Electrophoretic Expressed Protein in the Liver of Acute Alcoholic Abuse / 대한법의학회지

To study alcohol-related expressed proteins across postmortem livers, human liver tissues from two cadaveric subjects died of acute alcohol abuse and myocardialinfarct were compared. Liver tissues were separated by means of immobilized pH gradient- two-dimensional gel electrophoresis (2-DE) and Coomassie staining. Out of the differentially expressed proteins in 2-DE, four differential spots, which were strongly expressed in alcohol intoxication but faint in normal tissue, were selected and analyzed by matrix-assisted laser desorption/ionization time of flight mass spectrometry (MALDI-TOF-MS). Mass spectrometry (MS) analysis revealed these spots were mitochondrial aldehyde dehydrogenase precursor protein and 3-hydroxy-3-methylglutarylcoenzyme A synthase 2, respectively. Aldehyde dehydrogenase is the second-step enzyme for alcohol metabolism. 3-hydroxy-3-methylglutaryl-Coenzyme A synthase 2 is a key regulatory enzyme in the pathway for endogenous cholesterol synthesis. These preliminary data could suggest that the lipid metabolism-related protein as well as alcohol metabolic enzyme was expressed in acute alcohol abuse (binge drinking). Furthermore, it appears that proteomic tool would be applied for postmortem examination in the diagnosis of acute alcohol abuse

Ghrelin down-regulates ACAT-1 in THP-1 derived foam cells via growth hormone secretagogue receptor-dependent pathway / 中华心血管病杂志

<p><b>OBJECTIVE</b>To investigate the effects of Ghrelin on the expression of acyl coenzyme A:cholesterol acyltransferases-1 (ACAT-1) in THP-1 derived foam cells.</p><p><b>METHODS</b>The human monocytic leukemia cell line (THP-1) was chosen in our study. The differentiation of THP-1 cells into macrophages was induced by phorbol 12-myristate 13-acetate. Macrophages were then incubated with oxidized LDL (ox-LDL) to generate foam cells. Ghrelin and [D-Lys3]-GHRP-6, the special antagonist of growth hormone secretagogue receptor (GHS-R), were treated during foam cells formation. The ACAT-1 protein and mRNA levels were detected by Western blot and RT-PCR. The effect of variance of cholesterol content was measured by zymochemistry via-fluorospectrophotometer.</p><p><b>RESULTS</b>Ghrelin reduced the content of cholesterol ester in foam cells obviously. ACAT-1 protein and mRNA levels were also decreased. The antagonist of GHS-R inhibited the effects of Ghrelin on ACAT-1 expression in dose-dependent manner. The ACAT-1 mRNA levels of the GHS-R specific antagonist groups (10(-5), 5 x 10(-5), 10(-4) mol/L) were 1.14 +/- 0.04, 1.58 +/- 0.03, 2.40 +/- 0.16, significantly higher than that of the Ghrelin group (0.89 +/- 0.05). And the protein expressions were 1.25 +/- 0.09, 1.77 +/- 0.11, 2.30 +/- 0.09, also higher than that of the Ghrelin group (0.86 +/- 0.08).</p><p><b>CONCLUSIONS</b>Ghrelin might interfere atherosclerosis by down-regulating the expression of ACAT-1 via GHS-R pathway.</p>